CN106468689B - A kind of method of high performance liquid chromatography measurement gleptoferron content - Google Patents
A kind of method of high performance liquid chromatography measurement gleptoferron content Download PDFInfo
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- CN106468689B CN106468689B CN201610784087.5A CN201610784087A CN106468689B CN 106468689 B CN106468689 B CN 106468689B CN 201610784087 A CN201610784087 A CN 201610784087A CN 106468689 B CN106468689 B CN 106468689B
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- NFJRQODDTXZEBV-MXIFXDQUSA-M gleptoferron Chemical compound [Fe].[O-]O.OC[C@H]1O[C@H](OCC(O)C(O)C(O)C(O)C(O)C(O)=O)[C@H](O)[C@@H](O)[C@@H]1O NFJRQODDTXZEBV-MXIFXDQUSA-M 0.000 title claims abstract description 54
- 229950001273 gleptoferron Drugs 0.000 title claims abstract description 53
- 238000000034 method Methods 0.000 title claims abstract description 25
- 238000004128 high performance liquid chromatography Methods 0.000 title claims abstract description 17
- 238000005259 measurement Methods 0.000 title claims abstract description 16
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 18
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 8
- 229910000403 monosodium phosphate Inorganic materials 0.000 claims abstract description 7
- 235000019799 monosodium phosphate Nutrition 0.000 claims abstract description 7
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims abstract description 7
- 239000007788 liquid Substances 0.000 claims abstract description 6
- 238000000105 evaporative light scattering detection Methods 0.000 claims abstract description 4
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims abstract description 4
- 239000000377 silicon dioxide Substances 0.000 claims abstract description 4
- 239000000945 filler Substances 0.000 claims abstract description 3
- 239000000523 sample Substances 0.000 claims description 26
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims description 16
- 238000002347 injection Methods 0.000 claims description 10
- 239000007924 injection Substances 0.000 claims description 10
- 229910052742 iron Inorganic materials 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- 239000003643 water by type Substances 0.000 claims description 7
- 238000011088 calibration curve Methods 0.000 claims description 6
- 230000014759 maintenance of location Effects 0.000 claims description 6
- 238000010790 dilution Methods 0.000 claims description 5
- 239000012895 dilution Substances 0.000 claims description 5
- 239000000243 solution Substances 0.000 claims description 5
- 239000013558 reference substance Substances 0.000 claims description 4
- 239000012488 sample solution Substances 0.000 claims description 4
- 238000001914 filtration Methods 0.000 claims description 3
- 238000005070 sampling Methods 0.000 claims description 3
- 239000012086 standard solution Substances 0.000 claims description 3
- 239000011550 stock solution Substances 0.000 claims description 3
- IMNFDUFMRHMDMM-UHFFFAOYSA-N N-Heptane Chemical compound CCCCCCC IMNFDUFMRHMDMM-UHFFFAOYSA-N 0.000 claims 1
- 150000008065 acid anhydrides Chemical class 0.000 claims 1
- 150000002825 nitriles Chemical class 0.000 claims 1
- 238000011084 recovery Methods 0.000 abstract description 6
- 238000001514 detection method Methods 0.000 abstract description 2
- 230000035945 sensitivity Effects 0.000 abstract description 2
- 241001465754 Metazoa Species 0.000 description 4
- 208000015710 Iron-Deficiency Anemia Diseases 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 210000003743 erythrocyte Anatomy 0.000 description 3
- 239000007789 gas Substances 0.000 description 3
- -1 glucoheptose acid anhydride Chemical class 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 208000007502 anemia Diseases 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 238000002604 ultrasonography Methods 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000008857 Ferritin Human genes 0.000 description 1
- 238000008416 Ferritin Methods 0.000 description 1
- 108050000784 Ferritin Proteins 0.000 description 1
- 102000001554 Hemoglobins Human genes 0.000 description 1
- 108010054147 Hemoglobins Proteins 0.000 description 1
- 206010022971 Iron Deficiencies Diseases 0.000 description 1
- 241001071864 Lethrinus laticaudis Species 0.000 description 1
- 229910000805 Pig iron Inorganic materials 0.000 description 1
- 241000238370 Sepia Species 0.000 description 1
- 230000002180 anti-stress Effects 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 235000019441 ethanol Nutrition 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000012085 test solution Substances 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The present invention provides a kind of methods of high performance liquid chromatography measurement gleptoferron content, this method uses 1220 high performance liquid chromatograph of Agilent, using evaporative light scattering detector, 70 DEG C of drift tube temperature, gas flow rate 1.7L/min, 10 μ l of autosampler sample volume, include the following steps: (1) chromatographic condition: chromatographic column is the reverse-phase chromatographic column using octadecylsilane chemically bonded silica as filler, mobile phase is acetonitrile -0.5mol/L sodium dihydrogen phosphate, wherein, the volume ratio of acetonitrile and 0.5mol/L sodium dihydrogen phosphate is 26:74, flow velocity 1.0ml/min, 35 DEG C of column temperature.The method of high performance liquid chromatography measurement gleptoferron content of the present invention verifies method by precision test, linear relationship test and sample-adding recovery test etc., the results showed that this method is accurate and reliable, and detection range is big, and sensitivity and accuracy are high.
Description
Technical field
The invention belongs to herding fields, more particularly, to a kind of side of high performance liquid chromatography measurement gleptoferron content
Method.
Background technique
Iron is microelement necessary to animal growth, is the component part of a variety of enzymes in animal body.Pig iron-deficient
Anaemia is since iron deficiency causes red blood cell number in blood, blood erythrocyte concentration decline to pig during the growth process.The physiological property of pig
Determine that pig is the animal that iron-deficiency anemia most easily occurs in all animals.Pig can be caused when iron-deficiency anemia occurs
Blood oxygen carrying capacity wretched insufficiency, metabolic disturbance, anti-stress ability decline, to cause various diseases.Hypoferric anemia
The piglet that the day growth that mostly occurs is fast, individual is relatively strengthened.
Gleptoferron is preferably a kind of iron supplementary of clinical use effect, red blood cell after piggy injection gleptoferron
Number, hemoglobin, serum ferritin dramatically increase, and compared with general iron supplementary, gleptoferron absorbs fast, the utilization rate of iron
Height, injection volume is few, Small side effects, is the active drug for treating piglet iron-deficiency anemia.
Gleptoferron, English name: Gleptoferron, No. CAS be 57680-55-4, molecular formula C13H25FeO15,
Molecular weight 477.1741, brown to sepia crystalline powder, is soluble in hot water, does not dissolve in ethyl alcohol.Structural formula is shown in Fig. 1.
Summary of the invention
In view of this, the present invention is directed to propose a kind of high performance liquid chromatography measurement gleptoferron content method, efficiently,
Simplicity, high sensitivity, good separating effect, accuracy are high.
In order to achieve the above objectives, the technical scheme of the present invention is realized as follows:
A kind of method of high performance liquid chromatography measurement gleptoferron content, this method use the efficient liquid of Agilent1220
Chromatography, using evaporative light scattering detector, 70 DEG C of drift tube temperature, gas flow rate 1.7L/min, autosampler sample introduction
10 μ l are measured, are included the following steps:
(1) chromatographic condition: chromatographic column is the reverse-phase chromatographic column using octadecylsilane chemically bonded silica as filler, mobile phase
For acetonitrile -0.5mol/L sodium dihydrogen phosphate, wherein the volume ratio of acetonitrile and 0.5mol/L sodium dihydrogen phosphate is 26:
74, flow velocity 1.0ml/min, 35 DEG C of column temperature；
(2) Specification Curve of Increasing: precision weighs reference substance 25mg, is placed in 50ml brown volumetric flask, and it is pure that 40ml is added
Water ultrasound dissolves it all, is settled to scale after letting cool, and as gleptoferron standard solution stock solution, dilution is made step by step
Gleptoferron content is the solution of 5,10,20,50,100,200,500 μ g/ml, presses the chromatostrip of the step (1) respectively
Part measurement, 10 μ l of column chromatography column calculate separately the peak area under identical retention time, using content X as abscissa, with peak face
Product Y is ordinate, is made calibration curve equation Y=f (X), content is in 5-500 μ g/ml range；
(3) sample treatment: precision weighs that test sample is appropriate, and the solution that debita spissitudo is made is diluted with water, shakes up, with 0.45
μm organic filter filtering；
(4) content of gleptoferron in gleptoferron sample to be measured is detected: by treated glucoheptose acid anhydride to be measured
The chromatographic condition measurement of iron sample injection step (1) setting, 10 μ l of chromatographic column sample volume are calculated under identical retention time
Peak area, bring described step (2) the resulting calibration curve equation into, calculate machine gleptoferron sample solution to be measured
Concentration, then calculate by the sampling amount of sample the content of gleptoferron in sample.
Further, the chromatographic column selects Waters C18 column 4.6*250mm, 5 μm or Waters C18 column 4.6*
150mm, 5 μm；Preferably, chromatographic column selection Waters C18 column 4.6*250mm, 5 μm.
Compared with the existing technology, the method for high performance liquid chromatography of the present invention measurement gleptoferron content have with
Lower advantage:
The method of high performance liquid chromatography measurement gleptoferron content of the present invention is by precision test, linear pass
Method is verified in system's test and sample-adding recovery test etc., the results showed that this method is accurate and reliable, and detection range is big, sensitive
Degree and accuracy are high.
Detailed description of the invention
The attached drawing for constituting a part of the invention is used to provide further understanding of the present invention, schematic reality of the invention
It applies example and its explanation is used to explain the present invention, do not constitute improper limitations of the present invention.In the accompanying drawings:
Fig. 1 is the structural formula of gleptoferron；
Fig. 2 is that the HPLC of gleptoferron reference substance described in the embodiment of the present invention detects spectrogram；
Fig. 3 is the linear relationship chart and regression equation of gleptoferron of the present invention.
Specific embodiment
Unless otherwise stated, term used herein all has the meaning that those skilled in the art routinely understand, in order to
It is easy to understand the present invention, some terms used herein have been subjected to following definitions.
Below with reference to embodiment, the present invention will be described in detail.
A kind of method of high performance liquid chromatography measurement gleptoferron content, this method use the efficient liquid of Agilent1220
Chromatography, using evaporative light scattering detector, 70 DEG C of drift tube temperature, gas flow rate 1.7L/min, autosampler sample introduction
10 μ l are measured, are included the following steps:
(1) chromatographic condition:
Using octadecylsilane chemically bonded silica chromatographic column as chromatographic column, select Waters C18 column (4.6*250mm, 5 μm),
Mobile phase is -0.5% sodium dihydrogen phosphate of acetonitrile (V/V, 26:74), flow velocity 1.0ml/min, 35 DEG C of column temperature, using evaporative light
Scatter detector, 70 DEG C of drift tube temperature, gas flow rate 1.7L/min, 10 μ l of autosampler sample volume；
(2) drafting of standard curve:
Precision weighing precision weighs reference substance 25mg, is placed in 50ml brown volumetric flask, and 40ml pure water ultrasound, which is added, to be made
It is all dissolved, and is settled to scale after letting cool, and as gleptoferron standard solution stock solution, glucoheptose acid anhydride is made in dilution step by step
Iron content is the solution of 5,10,20,50,100,200,500 μ g/ml, be injected separately into step (1) setting into working condition
The 10 μ l of chromatographic column of HPLC device, calculates separately the peak area under identical retention time, using content X as abscissa, with peak area Y
For ordinate, calibration curve equation y=585588x-200000, R2=0.9995 are made, the results showed that gleptoferron content
Within the scope of 5-500 μ g/ml, peak area and concentration are in relation above；
(3) content of gleptoferron injection to be measured is detected:
Precision weighs gleptoferron injection to be measured, is dissolved in water, debita spissitudo is made, shake up, organic through 0.45 μm
Filter filtering.The 10 μ l of chromatographic column of the HPLC device into working condition of injection step (1) setting, calculates under retention time
Peak area brings step (2) resulting calibration curve equation into, calculates the concentration of machine gleptoferron sample solution to be measured,
The content of gleptoferron is calculated by the sampling amount of sample again, the calculation method of gleptoferron content is as follows:
Gleptoferron content (%)=gleptoferron to be measured sample solution concentration/(glucoheptose acid anhydride to be measured in sample
Iron samples weight * dilution gfactor) * 100%
Gleptoferron content/gleptoferron preparation labelled amount * 100% in gleptoferron (%)=sample
According to above formula, calculate that gleptoferron is labelled amount content 100.9% (dilution gfactor is sample extension rate
Inverse)；
(4) precision is investigated:
To same gleptoferron injection liquid samples, continuous sample introduction 6 times, peak area such as the following table 1:
The peak area of 1 gleptoferron injection liquid samples of table
| Number of injections | 1 | 2 | 3 | 4 | 5 | 6 |
| Peak area | 27272601 | 27274901 | 27266218 | 27273724 | 27265322 | 27261197 |
Peak area RSD is 0.02%, the results showed that, this HPLC method detects gleptoferron content precision height, reproducibility
It is good；
(5) it is loaded recovery test
The gleptoferron sample for accurately weighing known content is appropriate, then accurate a certain amount of gleptoferron of addition respectively
Sample 80%, 100%, 120% makes the gleptoferron concentration in test solution respectively in gleptoferron standard curve
Basic, normal, high region is measured in accordance with the law by operation of the invention, calculates the rate of recovery, and as a result average recovery rate is that 99.64%, RSD is
0.08%, show that this method sample recovery rate is good.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Within mind and principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (3)
1. a kind of method of high performance liquid chromatography measurement gleptoferron content, it is characterised in that: this method uses Agilent
1220 high performance liquid chromatographs, using evaporative light scattering detector, 70 DEG C of drift tube temperature, gas flow rate 1.7L/min, automatically
10 μ l of feeder amount, includes the following steps:
(1) chromatographic condition: chromatographic column is the reverse-phase chromatographic column using octadecylsilane chemically bonded silica as filler, and mobile phase is second
Nitrile -0.5mol/L sodium dihydrogen phosphate, wherein the volume ratio of acetonitrile and 0.5mol/L sodium dihydrogen phosphate is 26:74, stream
Fast 1.0ml/min, 35 DEG C of column temperature；
(2) Specification Curve of Increasing: precision weighs reference substance 25mg, is placed in 50ml brown volumetric flask, and it is super that 40ml pure water is added
Sound dissolves it all, is settled to scale after letting cool, and as gleptoferron standard solution stock solution, Portugal's heptan is made in dilution step by step
Sugared acid anhydride iron content is the solution of 5,10,20,50,100,200,500 μ g/ml, is surveyed respectively by the chromatographic condition of the step (1)
Fixed, 10 μ l of chromatographic column sample volume calculates separately the peak area under identical retention time, using content X as abscissa, with peak area Y
It for ordinate, makes calibration curve equation Y=f (X), content is in 5-500 μ g/ml range；
(3) sample treatment: precision weighs that test sample is appropriate, and the solution that debita spissitudo is made is diluted with water, shakes up, has with 0.45 μm
The filtering of machine filter head；
(4) content of gleptoferron in gleptoferron sample to be measured is detected: by treated gleptoferron sample to be measured
The chromatographic condition measurement of product injection step (1) setting, 10 μ l of chromatographic column sample volume calculate the peak under identical retention time
Area brings described step (2) the resulting calibration curve equation into, calculates the dense of machine gleptoferron sample solution to be measured
It spends, then calculates the content of gleptoferron in sample by the sampling amount of sample.
2. the method for high performance liquid chromatography measurement gleptoferron content according to claim 1, it is characterised in that: described
Chromatographic column select Waters C18 column 4.6*250mm, 5 μm or Waters C18 column 4.6*150mm, 5 μm.
3. the method for high performance liquid chromatography measurement gleptoferron content according to claim 2, it is characterised in that: described
Chromatographic column select Waters C18 column 4.6*250mm, 5 μm.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4493826A (en) * | 1982-01-25 | 1985-01-15 | Schering Corporation | Gentamicin-gleptoferron compositions |
| CN1538839A (en) * | 2001-07-31 | 2004-10-20 | ��ī�����˹��ʽ���� | Swine growth promoters and method of promoting swine growth |
| KR20130052329A (en) * | 2011-11-11 | 2013-05-22 | 주식회사 단바이오텍 | Iron supplement for oral administration |
-
2016
- 2016-08-31 CN CN201610784087.5A patent/CN106468689B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4493826A (en) * | 1982-01-25 | 1985-01-15 | Schering Corporation | Gentamicin-gleptoferron compositions |
| CN1538839A (en) * | 2001-07-31 | 2004-10-20 | ��ī�����˹��ʽ���� | Swine growth promoters and method of promoting swine growth |
| KR20130052329A (en) * | 2011-11-11 | 2013-05-22 | 주식회사 단바이오텍 | Iron supplement for oral administration |
Non-Patent Citations (2)
| Title |
|---|
| Iron methionine as a source of iron for the neonatal pig;E.B. Kegley 等;《Nutrition Research》;20021031;第22卷(第10期);第1209-1217页 |
| 用葡庚糖酐铁与葡聚糖铁治疗仔猪缺铁性贫血;山田裕 等;《日本兽医师会杂志》;19861231;第39卷(第11期);第693页 |
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